ANTICANCER RESEARCH 35: 4625-4632 (2015) WNT16-expressing Acute Lymphoblastic Leukemia Cells are Sensitive to Autophagy Inhibitors after ER Stress Induction MELETIOS VERRAS1, IOANNA PAPANDREOU2 and NICHOLAS C. DENKO2 1General Biology Laboratory, School of Medicine, University of Patra, Rio, Greece; 2Department of Radiation Oncology, Wexner Medical Center and Comprehensive Cancer Center, The Ohio State University, Columbus OH, U.S.A. Abstract. Background: Previous work from our group showed burden of proteins in the ER through decreased translation, hypoxia can induce endoplasmic reticulum (ER) stress and increased chaperone expression, and increased removal of the block the processing of the WNT3 protein in cells engineered malfolded proteins through degradation. If the cell is unable to express WNT3a. Acute lymphoblastic leukemia (ALL) cells to relieve the ER stress, then cellular death can ensue (3). with the t(1:19) translocation express the WNT16 gene, which The microenvironment of solid tumors is often poorly is thought to contribute to transformation. Results: ER-stress perfused, resulting in regions of hypoxia and nutrient blocks processing of endogenous WNT16 protein in RCH-ACV deprivation (4, 5). However, hypoxia has been also shown to and 697 ALL cells. Biochemical analysis showed an impact cancer of the bone marrow such as aggressive aggregation of WNT16 proteins in the ER of stressed cells. leukemia (6). In addition to inducing the hypoxia-inducible These large protein masses cannot be completely cleared by factor 1 (HIF1) transcription factor, severe hypoxia induces ER-associated protein degradation, and require for additional stress in the ER (7, 8). Cells with compromised ability to autophagic responses. Pharmacological block of autophagy respond to ER stress (due to genetic ablation of the x-box significantly increased cell death in ER-stressed ALL. binding protein (XBP1) transcription factor or the Protein Furthermore, murine cells engineered to express WNT16 are kinase RNA- like endoplasmic reticulum kinase (PERK)) similarly sensitized. Conclusion: ALL cells expressing WNT16 grow poorly as model tumors (9, 10). Pharmacological are sensitive to ER stress, and show enhanced killing after inhibition of ER stress responses can also have anticancer addition of chloroquine. These findings suggest a potential effects in vitro and in model myeloma (11). Hypoxia-induced clinical application of inducers of ER stress with inhibitors of ER stress can inhibit the secretion and paracrine activity of autophagy in patients with high-risk ALL. growth factors such as WNT family members (12). WNT proteins have highly conserved series of 25-27 cysteine Environmental conditions or drugs can interfere with the residues that are thought to establish a complex tertiary proper functioning of the endoplasmic reticulum (ER) (1). A structure essential for their activity (13). Lack of oxygen block to the normal flow of proteins through the ER results in disrupts the normal formation of disulphide bonds in the ER stress and elicits the compensatory unfolded protein WNT proteins, leading to their retention in the ER and response (UPR) (2). Proteins can accumulate in the ER in an ultimately in their degradation through proteasomal and improperly folded state after a block of essential ER functions autophagic mechanisms (12). Activation of autophagy under such as protein folding, disulfide bond formation, or ER stress conditions is therefore compensatory and leads to glycosylation. Due to the presence of unfolded proteins in the the degradation of unfolded/misfolded protein aggregates ER, cells initiate a series of responses designed to reduce the that are not soluble and cannot be degraded by ER associated degradation (ERAD) (14, 15). We have previously shown that hypoxia induces autophagy via AMP activated protein kinase (AMPK) Correspondence to: Nicholas C. Denko, Department of Radiation activity, in an HIF-independent process (16). We have also Oncology, Wexner Medical Center and Comprehensive Cancer shown that hypoxic ER stress can inhibit the processing of Center, Ohio State University, Columbus OH 43210, U.S.A. E-mail: the WNT family of secreted glycoproteins in engineered [email protected] cancer cells (12). In the present study, we again use WNT Key Words: Unfolded protein response, tumor hypoxia, acute glycoproteins as tools to explore the hypothesis that lymphoblastic leukemia, WNT growth factors, ER-associated autophagy is integral to the hypoxia-induced ER stress degradation. response. Here we report studies examining expression of 0250-7005/2015 $2.00+.40 4625 ANTICANCER RESEARCH 35: 4625-4632 (2015) endogenous WNT16 protein in pre-B acute lymphoblastic Results leukemia (ALL) cell lines after treatment with conditions that induce ER stress. Expression of WNT16 is compromised by ER stress in ALL cells. Previous work from our group examined the processing Materials and Methods of WNT3A in cells engineered to express the protein after ER stress. We engineered murine L cells and human RKO colorectal cancer cells to express exogenous WNT3A and Cells, cell culture and reagents. ProB leukemic cell lines RCH-ACV and 697 cells were cultured in RPMI with 20% fetal bovine serum found that WNT processing was impaired during ER stress (FBS), Murine fibroblast L cells were cultured in Dulbecco’s from hypoxia, and this down-regulated β-catenin signaling. modified eagle medium (DMEM) with 10% FBS. For moderate We now wished to extend these studies to determine if tumor hypoxia, cells were treated in a variable-oxygen Invivo2 humidified cells that naturally expressed WNT family members were hypoxia workstation (Ruskinn Technologies, Bridgend, UK). Severe also sensitive to ER stress. Pre-B ALL cells that contain the hypoxia was generated in an anaerobic workstation gassed with 5% fusion protein which joins the transactivation domain of the CO , 5% H and 95% N containing a palladium catalyst (Sheldon 2 2 2 E2A protein with the DNA binding domain of the Co., Cornelius, OR, USA). Transient and stable transfections were performed using Lipofectamine (Invitrogen, Carlsbad, CA, USA). homeoprotein PBX express WNT16 due to the activity of the MG-132, tunicamycin, thapsigargin, chloroquine, E64 and pepstatin fusion protein (17, 18). We therefore obtained RCH-ACV were purchased from Sigma-Aldrich (Saint Louis, MO, USA). and 697 ALL cells and examined WNT16 expression and Dithiothreitol (DTT) was purchased from Invitrogen. The pLPC- biological activity by western blot after treatment with ER Wnt16 and pLPC empty retroviral vectors were a kind gift from Dr. stressing agents. Figure 1 shows that treatment with reduced Amato Giaccia. oxygen tension causes reduced expression of endogenous WNT16 in both cell lines (Figure 1A and B). Furthermore, Retroviral transduction. WNT16 expressing cells were generated by retroviral transduction. A WNT16-expressing retroviral vector we also show that there is decreased WNT signaling because (pLPC-WNT16) was transfected into HEK 293 Phoenix cells using there is less stabilized β-catenin in these cells after treatment Lipofectamine as directed by the manufacturer (Life Technologies, with hypoxia (Figure 1A and B). Grand Island NY USA). After 48 h, the supernatant containing the We confirmed that the decreased WNT16 protein is due to retrovirus was collected, filtered and used to transduce the indicated incomplete processing in the ER using the thiols protection cell lines in the presence of 5 μg/ml polybrene (Sigma Aldrich, St assay. Extracts were collected in N-ethylmaleimide and this Louis MO, USA). WNT16-positive clones were selected using stabilized the thiols and disulphide bonds in the WNT16 puromycin, and expression confirmed by immunoblot. protein. The samples were electrophoresed either on a non- Western blotting. In brief, treated cells were harvested in RIPA buffer, reducing (Figure 1C left) or reducing (Figure 1C right) SDS lysates were sonicated, cleared by centrifugation, and protein polyacrylamide gel. Figure 1C shows that without addition concentrations were quantitated by BCA reagent (Life Technologies, of DTT to break disulphide bonds, a high fraction of the Grand Island NY USA). Proteins (25–50 μg) were electrophoresed on WNT16 proteins extracted from hypoxia-treated cells a reducing Tris-Tricine gel, and electroblotted to polyvinyl difluoride migrate as very large protein aggregates with apparent membrane. Antibodies used were mouse anti-β-catenin (BD velocity indicating sizes above 250 kDa. Addition of DTT to Biosciences Pharmingen San Diego CA USA), mouse anti-human WNT16 (BD Biosciences Pharmingen), LC3 (MBL International these extracts breaks all disulfide bonds and collapses the Woburn MA USA), and mouse anti-β-actin (Abcam Hong Kong). WNT16 reactive material to an apparent monomeric size of Primary antibodies were detected with species-specific horseradish 45 kDa. We interpret these results to indicate that severe peroxidase secondary antibodies (DAKO Carpenteria CA USA) and hypoxia interferes with correct disulphide bond formation of visualized with ECL (Amersham Piscataway NJ USA) on a Storm 860 WNT16 proteins in the ER, because oxygen is the terminal phosphoimager (Molecular Devices San Francisco CA USA). electron acceptor in the reaction forming disulphide bonds Thiol modification blocking assay (treatment with N-ethylmaleimide). (7). Without correct 3-dimensional structure, WNT proteins Cells were cultured for 24 h and then lysed in RIPA buffer (1% aggregate, and this signals for